Burkholderia pseudomallei, the etiologic agent of melioidosis, is a significant cause of morbidity and mortality in humans. The closely related pathogen, B. mallei, causes equine glanders and can also afflict humans. Due to the high lethality of these infections, the paucity of effective antibiotics or vaccines, and concerns regarding the potential use of these pathogens as biological weapons, understanding virulence mechanisms and developing means to protect populations at risk are high priorities. Cell-cell spread is a hallmark of Burkholderia pathogenesis. We have shown that efficient cell-cell spread is critically dependent on the ability of the bacterium to invade eukaryotic cells, escape from endosomes, move intracellularly and fuse plasma membranes, forming multinucleated cells (MNCs). We have discovered that cell fusion represents the primary means of intercellular spread during infection of cell monolayers in vitro, and we have proposed a model that is similar to mechanisms used by enveloped viruses. Studies in our laboratory implicate a type VI secretion system (T6SS-5) as a key determinant in this process, and VgrG5, the trimeric tip of the T6SS-5 apparatus. There remain significant unknowns regarding the mechanism of cell fusion and other critical intracellular lifecycle steps that promote cell-cell spread, including th full inventory of bacterial and host factors that participate in these processes. The purpose of this project is to develop novel therapeutic strategies for pathogenic Burkholderia species and advance our understanding of critical factors that participate in pathogenesis. Specifically, we propose to 1) identify small molecule inhibitors of cell-cell spread, and 2) identify critical bacterial factors that are required for the intercellular lifecycle. For small molecules found to effectively inhibit cell-cell spread, we have designed a systematic series of follow-up experiments to pinpoint the lifecycle stage that has been disrupted and determine the mechanism of action and molecular target. Independent and complementary approaches will dissect the role of newly identified bacterial factors in membrane fusion and intercellular spread and assess the potential of targeting these proteins for therapeutic intervention.